Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Citation: Garcia, B. L., Zhi, H., Wager, B., Hook, M., & Skare, J. T. (2016). Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex. Plos Pathogens, 12(1), 28. doi:10.1371/journal.ppat.1005404Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems

Pure chemotaxis of non-adherent neutrophils toward <i>C</i>. <i>posadasii</i>.

<p>Using micropipettes, endospores (A) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s001" target="_blank">S1 Video</a>) and spherules (B) are maneuvered to different positions relative to the cell without touching the cell. In this configuration, chemotaxis takes the form of a directional, protrusive pseudopod extended by the neutrophil toward the target. The relative times of all video images are included. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.t001" target="_blank">Table 1</a> summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.</p

Phagocytosis of <i>C</i>. <i>posadasii</i> endospores by initially passive human neutrophils.

<p>(See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s002" target="_blank">S2 Video</a>.) Four example experiments are presented as vertical filmstrips. The experiment buffer contained 10% heat-treated autologous serum, which prevented chemotaxis (such as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.g003" target="_blank">Fig 3</a>) but not the engulfment of the endospore after direct contact with the neutrophil surface. The relative times of all video images are included. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.t001" target="_blank">Table 1</a> summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.</p

Comparison of the behavior of neutrophils from patients with chronic coccidioidomycosis and from healthy donors.

<p>A. Filmstrips illustrate the responses of neutrophils from the two donor groups to contact with <i>C</i>. <i>posadasii</i> endospores and antibody-coated beads, respectively (in the presence of autologous serum). All scale bars denote 10 μm. B. Results of the quantitative analysis of the positional trajectories (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.g007" target="_blank">Fig 7</a>) of <i>C</i>. <i>posadasii</i> endospores and antibody-coated beads during phagocytosis by neutrophils from the two donor groups. (Error bars denote standard deviations, and asterisks mark differences that are statistically significant. The number <i>N</i> of analyzed single-cell experiments is included in the figure.).</p

Overview of single-cell experiments.

<p>A. Schematic of our dual-micropipette manipulation system. The chamber volume is created by trapping buffer solution between two horizontal microscope coverslips. Facing pipettes access this volume through the chamber's two open sides. Vertically movable water reservoirs allow us to control the pipette-aspiration pressure with high resolution. The aspiration pressure of the right pipette is monitored in real time by measuring the height difference between the main reservoir (which is connected to the pipette) and a pre-zeroed reference reservoir. The included example videomicrographs demonstrate how micropipettes are used to pick up individual targets (B) and neutrophils (C) with gentle suction. After lifting these objects above the chamber bottom, they can be maneuvered in 3D to set up experiments that assess target recognition either from a distance (D) or upon direct physical contact (E). All scale bars denote 10 μm.</p

Analysis of the positional trajectory of a target particle (here: a <i>C</i>. <i>posadasii</i> endospore) during phagocytosis by a pipette-held neutrophil.

<p>The annotated videomicrographs at the top demonstrate our measurement of the distance between the center of the target particle and the opposite side of the main cell body (<i>red straight line</i>). This distance (relative to its initial value) is plotted as a function of time in the bottom graph (<i>red curve</i>). Numbered circles correspond to the time points at which the respective example images were taken. This type of graph allowed us to determine the maximum push-out distance as well as the pull-in speed of the target as shown. The engulfment time (defined in the text) was found by inspection of the recorded video images.</p

Phagocytosis of <i>C</i>. <i>posadasii</i> spherules by initially passive human neutrophils.

<p>(See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s003" target="_blank">S3 Video</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s004" target="_blank">S4</a> Video.) Four example experiments are presented as vertical filmstrips. (The spherule in the first panel is immature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.ref071" target="_blank">71</a>].) The experimental conditions were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.g004" target="_blank">Fig 4</a>. The relative times of all video images are included. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.t001" target="_blank">Table 1</a> summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.</p

Overview of <i>Coccidioides</i> spp. and human neutrophils.

<p>A. Endemic areas of <i>Coccidioides</i> spp. B. Life cycle of <i>Coccidioides</i> spp. Vegetative mycelia exist in the soil and produce arthrospores during periods of low precipitation. Following aerosolization and inhalation of arthrospores, immature spherules develop and transition into large spherules containing hundreds of endospores. The mature spherules eventually rupture and release the endospores, reinitiating the spherule/endospore phase. (Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.ref012" target="_blank">12</a>] with permission.). C. Composite videomicrographs of typical <i>C</i>. <i>posadasii</i> endospores (<i>top</i>) and spherules (<i>bottom</i>). D. Composite brightfield videomicrographs of quiescent human neutrophils as used in the experiments. E. H&E-stained human neutrophils after neutrophil enrichment. All images in C-E are shown at the same magnification (some cell shrinkage occurred during H&E-staining). The common scale bar denotes 10 μm.</p

Frustrated phagocytosis of <i>C</i>. <i>posadasii</i> spherules by multiple neutrophils.

<p>A. Shortly after manipulating an immature spherule and a first neutrophil (#1) into contact, a second neutrophil (#2) touches and adheres to the spherule by chance. Both cells proceed to spread over the surface of the spherule. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s005" target="_blank">S5 Video</a>.) B. Using micropipettes, three neutrophils are sequentially brought into contact with the same spherule and proceed to attack it. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129522#pone.0129522.s006" target="_blank">S6 Video</a>.) The relative times of all video images are included. C,D. Bulk assay to verify the recognition of live <i>C</i>. <i>posadasii</i> spherules by human neutrophils. The spherules were incubated with neutrophils for 5 min. (C) or for 20 min. (D) in suspension with gentle mixing on a rotator, then fixed and H&E-stained. Arrows point to particularly spread-out leukocytes. (Some cell shrinkage occurred during H&E-staining.) All scale bars denote 10 μm.</p

Quantitative analysis of the time course of phagocytosis of <i>C</i>. <i>posadasii</i> endospores (<i>left</i>) and spherules (<i>right</i>) by human neutrophils.

<p>Representative timelines of the target position (aligned for best overlap of the trajectories during the pull-in phase) (A), the cell-surface area (B), and the cortical tension (C) are shown for three particles of each type. For each target type, a given color indicates the same cell-target pair throughout parts A, B, and C. The three phases identified at the bottom of the figure were determined by inspection of the time-dependent neutrophil morphologies in the recorded image sequences. Positive values of the target position shown in part A reflect a push-out of the particle. A monotonous decrease of the position values characterizes the pull-in phase. The end of the pull-in phase marks the start of the final phase. The inset in the right panels depicts the cell behavior over an extended period of time (~33 minutes). Common axis titles are shown only once at the left and bottom of the figure.</p